Immunoelectron Microscopy of Amyloid Fibrils

3 μM of recombinant Aβ42 (Walsh et al., 2009 (link)) with 0.7 equivalents of recombinant BRICHOS (Willander et al., 2012a (link)) were incubated at 37°C overnight. Aliquots of 2 μl were loaded on Nickel-coated grids, and excess of sample was removed. The grids were placed on a drop of 1% BSA in TBS, incubated for 30 minutes at RT, and then washed for 10 minutes on each of three drops of TBS. The grids were then placed on drops of rabbit anti-C-terminal proSP-C antibody diluted 1:200 in TBS, and incubated overnight at +4°C. After washing 10 minutes on each of five drops, the grids were placed on a drop of goat anti-rabbit IgG coupled to 10-nm gold particles diluted 1:40 in TBS, and incubated for 2 hours at RT. The grids were then washed five times as before, followed by negative staining with 2% uranyl acetate in 50% ethanol. The immunolabeled fibrils were examined using a Hitachi H7100 TEM operated at 75 kV.